APOBEC3 (A3) proteins are restriction factors that provide innate immunity against HIV-1 in the absence of viral infectivity factors (Vif). These single-stranded DNA- (ssDNA) and RNA-binding proteins are active deoxycytidine deaminases. Their ability to restrict of HIV replication is primarily linked to C ? U deamination of the minus-strand of DNA copy of viral RNA, resulting in a G ? A mutation in the viral DNA genome and functional inactivation of the virus. Despite the importance of A3G in HIV restriction, knowledge regarding the molecular mechanisms that underlie the interactions of A3 with nucleic acids is very limited. The objective of this application is to unravel mechanisms of A3-ssDNA interactions and the features of A3G-ssRNA that define A3G?s incorporation in virions. Our ultimate goal is to translate this knowledge toward the prevention and treatment of HIV. Our central hypothesis is that the two-domain property of A3G and A3F proteins is critical for maintaining all aspects of A3 antiviral activity and encapsidation in virions. Our rationale is that understanding the fundamental mechanisms of A3-ssDNA and A3-RNA interactions will guide the development of approaches to control and inhibit virus replication. To accomplish this goal and test our hypothesis, we will use a set of the nanoimaging and probing tools and develop a nanoarray approach that allows us to assemble A3 proteins in oligomers with defined sizes. Guided by strong preliminary data, we will test our major hypothesis through the following three specific aims: Specific Aim 1: Reveal the interplay between the catalytic and DNA-binding domains of A3 proteins. Hypothesis: The non-catalytic DNA-binding domain defines the complex assembly and modulates the deamination activity of A3 proteins. Specific Aim 2: Identify the role of A3G and A3F oligomerization in interactions with DNA. Hypothesis: Oligomerization increases deamination activity and stability of A3 proteins in complexes with ssDNA. Specific Aim 3: Characterize the interaction of A3G and A3F with RNA. Hypothesis: Oligomerization of A3 proteins is a critical for interaction with viral RNA and encapsidation in virions. These studies are expected to lead to the development of new and innovative preventative strategies and treatments for HIV. The proposed study is innovative in that it presents a novel approach to study A3-DNA and RNA complexes and develops a set of new nanotechnology methods with broad biomedical applications. The proposed research is significant in that the findings will lay the foundation for further improving the innate immunity property of A3. Additionally, A3 oligomers can avoid Vif-dependent degradation. Thus, the availability of A3 oligomers of select sizes assembled as nanoarrays opens prospects for testing these nanoassemblies as a means of HIV prevention.